Coordinated changes in gene expression are required for the correct development of both heart and skeletal muscle. The myoD family of myogenic factors, by binding to E boxes in the regulatory region of muscle genes, appears to control expression of muscle genes during skeletal myogenesis. However, not all muscle genes required E boxes for transcriptional activity and cardiac cells do not express any of the myogenic factors. Thus, alternative regulatory mechanism and transcription factors must exist. The factor which regulates the expression of the cardiac troponin T (cTNT) gene appears to be one such alternate transcription factor. Transcription of the cTNT gene in embryonic heart and skeletal muscle is not dependent on E-box sequences but is absolutely dependent on the presence of two copies of a M-CAT motif (5'-CATTCCT-3'). This motif binds a factor termed the M-CAT binding factor (MCBF). MCBF is also required for high level muscle- specific expression of several other muscle genes in heart and skeletal muscle cells. Thus, the M-CAT motif and its cognate transcription factor represents a muscle gene transcriptional system operating in heart and skeletal muscle that is different from the myoD regulatory loop. Two CDNAs encoding chicken MCBF have been cloned. One of these is a transcription activator. Sequence comparison with other proteins shows that MCBF is highly homologous to the SV40 transcription enhancer factor, TEF-1. MCBF and TEF-1 belong to the TEA/ATTS family of proteins whose members have been implicated in developmental and transcriptional control from plant to man. These proteins do not have obvious structural features more commonly seen in other well-characterized transcription factors. Therefore, the overall goal of this research is to determine how MCBF functions to activate the cTNT gene promoter in muscle cells. To begin to achieve this goal, the following experimental approaches will be used: 1) The expression pattern of the MCBFs during heart and skeletal muscle development will be analyzed to determine how it relates to cTNT expression. 2) To determine how MCBF interacts with the M-CAT motif, the physical state of MCBF in solution and upon DNA binding will be determined; and then the protein domains essential for DNA binding, activation, and interaction will be defined by a combination of mutational studies and in vitro/in vivo analyses. 3) The MCBF/M-CAT interactions occurring at the cTNT promoter essential for transcriptional activation will be defined by analysis of the affinity and binding characteristics of MCBF to the two cTNT M-CAT sites using gel shift and methylation interferences assays with wildtype and mutant cTNT promoters and MCBF proteins. Protein-protein interactions will be studied by cross-linking and immunoprecipitation. Results of these studies will lead not only to a better understanding of muscle gene regulation but also how members of the TEA/ATT family interact to control different development and transcriptional processes.